1. Field of the Invention
The field of this invention relates to methods of controlling the tumorigenicity and/or metastasis of neoplastic cells. Specifically it relates to the use of antisense sequences directed against the R1 and R2 components of mammalian ribonucleotide reductase.
2. State of the Art
The first unique step leading to DNA synthesis is the conversion of ribonucleotides to their corresponding deoxyribonucleotides, a reaction that is catalyzed in a cell cycle specific manner by the housekeeping gene ribonucleotide reductase [Lewis et al., 1978; Reichard, 1993; Wright, 1989a; Wright et al., 1990a; Stubbe, 1989]. The mammalian enzyme is composed of two dissimilar dimeric protein components often called R1 and R2, which are encoded by two different genes located on different chromosomes [Bjorklund et al., 1993; Tonin et al., 1987]. Mammalian protein R1 is a homodimeric structure, with a molecular weight of about 170 kDa, and has substrate sites and allosteric effector sites that control enzyme activity and substrate specificity [Wright, 1989A; Thelander et al., 1980; Caras et al., 1985; Wright et al., 1990a]. Protein R2 is a homodimer, with a molecular weight of 88 kDa, and forms two equivalent dinuclear iron centers that stabilizes a tyrosyl free radical required for catalysis [Wright et al., 1990a; Thelander et al., 1985; McClarty et al., 1990]. R1 and R2 proteins interact at their C-terminal ends to form an active holoenzyme [Reichard, 1993; Wright et al., 1990a; Davis et al., 1994].
R1 and R2 are differentially regulated during the cell cycle. There is an S-phase correlated increase in the R2 protein resulting from its de novo synthesis [Lewis et al., 1978; Mann et al., 1988]. The activity of ribonucleotide reductase, and therefore DNA synthesis and cell proliferation, is controlled in proliferating cells during the cell cycle by the synthesis and degradation of the R2 component [Eriksson et al., 1984]. The rate-limiting R2 component is a phosphoprotein capable of being phosphorylated by the CDC2 and CDK2 protein kinase mediators of cell cycle progression [Chan et al., 1993], and contains non-heme iron that stabilizes an unique tyrosyl free radical required for enzyme activity [Reichard, 1993; McClarty et al., 1990.
The levels of the R1 protein do not appear to change substantially during the cell cycle of proliferating cells and can be detected throughout the cell cycle. Synthesis of R1 mRNA, like R2 mRNA appears to occur mainly during S phase [Eriksson et al., 1984; Choy et al., 1988; Mann et al., 1988]. The broader distribution of the R1 protein during the cell cycle is attributed to its longer half life as compared to the R2 protein [Choy et al., 1988; Mann et al., 1988].
Regulation of ribonucleotide reductase, and particularly the R2 component, is altered in malignant cells exposed to tumor promoters or to the growth factor TGF-xcex2 [Amara, et al., 1994; Chen et al., 1993; Amara et al., 1995b; Hurta and Wright, 1995A; Hurta et al., 1991]. Higher levels of enzyme activity have been observed in cultured malignant cells when compared to nonmalignant cells [Weber, 1983; Takeda and Weber, 1981; Wright et al., 1989a], and increased levels of R2 protein and R2 mRNA have been found in pre-malignant and malignant tissues as compared to normal control tissue samples [Saeki et al., 1995; Jensen et al., 1994].
Compounds like hydroxyurea inhibit ribonucleotide reductase activity by destabilizing the iron center of the R2 protein causing the destruction of the tyrosyl free radical [McClarty et al., 1990], and preventing cells from progressing through S-phase of the cell cycle [Ashihara and Baserga, 1979].
Breakthroughs in molecular biology and the human genome project have opened previously unforeseen possibilities for targeted intervention with mammalian gene expression [Blaese, 1997]. These include approaches such as disruption of specific genes. Antisense (AS) oligonucleotides (AS-ON) designed to hybridize with specific sequences within a targeted mRNA are one example of such targeted intervention. In general, antisense oligonucleotides interact well with phospholipid membranes [Akhter et al., 1991]. Following their interaction with the cellular plasma membrane, they may be actively, or passively, transported into living cells [Loke et al., 1989], and this may occur by a saturable mechanism predicted to involve specific receptors [Yakubov et al., 1989].
Many excellent reviews have covered the main aspects of antisense technology and its enormous therapeutic potential. There are reviews on the chemical [Crooke, 1995], cellular [Wagner, 1994] and therapeutic [Hanania, et al., 1995; Scanlon, et al., 1995; Gewirtz, 1993] aspects of this rapidly developing technology. Within a relatively short time, ample information has accumulated about the in vitro use of antisense oligonucleotides in cultured primary cells and cell lines as well as for in vivo administration of such oligonucleotides for suppressing specific processes and changing body functions in a transient manner. Further, enough experience is now available in vitro and in vivo in animal models to predict human efficacy.
It would be useful to have antisense oligonucleotides available to control tumorigenicity and/or metastatic potential in premalignant or malignant cell wherein the R1 and R2 components of ribonucleotide reductase were utilized.
The present invention provides an isolated antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R1 or sequence segment thereof. This sequence may further comprise a reduced dimer formation and reduced self-complementary interactions.
Another aspect of this invention is a synthetic antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R2 or sequence segment thereof. This sequence may further comprise a reduced dimer formation and reduced self-complementary interactions.
Also provided is a a pharmaceutical composition for inhibiting tumor cell growth in a mammal comprising an effective amount of the antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R1 or sequence segment thereof and a pharmaceutically acceptable carrier or diluent.
Also provided is a pharmaceutical composition for inhibiting tumor cell growth in a mammal comprising an effective amount of the antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R2 or sequence segment thereof and a pharmaceutically acceptable carrier or diluent.
In one of its method aspects, this invention provides a method of inhibiting the tumorigenicity of neoplastic cells in a mammal which method comprises contacting the neoplastic cell with an effective amount of at least one antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence selected from the group consisting of sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R1 or sequence segment thereof or sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R2 or sequence segment thereof.
Another aspect is a method of inhibiting the tumorigenicity of neoplastic cells resistant to chemotherapeutic drugs in a mammal which method comprises identifying patients who have tumors that are resistant to a chemotherapeutic drug; and contacting the tumor with the chemotherapeutic drug to which the tumor is resistant and an antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence selected from the group consisting of sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R1 or sequence segment thereof or sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R2 or sequence segment thereof wherein the amount of the chemotherapeutic drug and the antisense oligonucleotide is sufficient to inhibit tumor cell growth. The amount of antisense oligonucleotide alone may be insufficient to inhibit tumor cell growth.
Another aspect is a method of increasing sensitivity of neoplastic cells to chemotherapeutic drugs in a mammal by contacting the tumor with an antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence selected from the group consisting of sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R1 or sequence segment thereof or sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R2 or sequence segment thereof.
Another aspect is a method of inhibiting metastasis of tumor cells in a mammal which method comprises administering to said mammal an amount sufficient to inhibit tumor cell growth of an antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence selected from the group consisting of sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R1 or sequence segment thereof or sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R2 or sequence segment thereof.
Another aspect is an isolated DNA with a sequence comprising a transcriptional initiation region and a sequence encoding an antisense oligonucleotide from at least about three nucleotides or nucleotide analogues to about fifty nucleotides in length comprising a sequence selected from the group consisting of sequences complementary to a mRNA sequence of a mammalian ribonucleotide reductase protein component R1 or sequence segment thereof or sequences complementary to a mRNA sequence of a mammalian ribonucleotide eductase protein component R2 or sequence segment thereof.